Antisense-induced ribosomal frameshifting

Henderson, Clark M.; Anderson, Christine B.; Howard, Michael T.

Surprisingly, the addition of AZ1A (0 spacer) also induced high-level frameshifting into the À1 reading frame in a manner which was modestly inhibited by the addition of spermidine (19% in the absence and 10% in the presence of 0.4 mM exogenous spermidine) ( Figure 3A and Supplementary Table 2 ). No À1 frameshift product was observed when the wild-type antizyme cassette was examined in the absence of antisense oligonucleotide addition (Figure 2 ; AZwt). As the AZ1A antisense oligonucleotide was designed to anneal directly adjacent to the UGA codon of the shift site, it was of interest to determine whether the wild-type antizyme pseudoknot could induce À1 frameshifting when located in the equivalent position. To address this, a new construct p2luc-AZ1-0sp ( Figure 1A ) was made by deleting the 3 nt spacer between the pseudoknot and the shift site of p2luc-AZ1wt. In this case, the wild-type pseudoknot is directly 3 0 adjacent to the shift site. The products of in vitro transcription and translation were separated by SDS-PAGE. No À1 frameshift product was observed and levels of the +1 frameshift product were significantly reduced to $3% ( Figure 3D and Supplementary Table 2) .